Intrasarcoplasmic Amyloidosis Impairs Proteolytic Function of Proteasomes in Cardiomyocytes by Compromising Substrate Uptake

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1 Intrasarcoplasmic Amyloidosis Impairs Proteolytic Function of Proteasomes in Cardiomyocytes by Compromising Substrate Uptake Quanhai Chen,* Jin-Bao Liu,* Kathleen M. Horak, Hanqiao Zheng, Asangi R.K. Kumarapeli, Jie Li, Faqian Li, A. Martin Gerdes, Eric F. Wawrousek, Xuejun Wang Abstract The presence of increased ubiquitinated proteins and amyloid oligomers in failing human hearts strikingly resembles the characteristic pathology in the brain of many neurodegenerative diseases. The ubiquitin proteasome system (UPS) is responsible for degradation of most cellular proteins and plays essential roles in virtually all cellular processes. UPS impairment by aberrant protein aggregation was previously shown in cell culture but remains to be demonstrated in intact animals. Mechanisms underlying the impairment are poorly understood. We report here that UPS proteolytic function is severely impaired in the heart of a mouse model of intrasarcoplasmic amyloidosis caused by cardiac-restricted expression of a human desmin related myopathy-linked missense mutation of B-crystallin (CryAB R120G ). The UPS impairment was detected before cardiac hypertrophy, and failure became discernible, suggesting that defective protein turnover likely contributes to cardiac remodeling and failure in this model. Further analyses reveal that the impairment is likely attributable to insufficient delivery of substrate proteins into the 20S proteasomes, and depletion of key components of the 19S subcomplex may be responsible. The derangement is likely caused by aberrant protein aggregation rather than loss of function of the CryAB gene because UPS malfunction was not evident in CryAB-null hearts and inhibition of aberrant protein aggregation by Congo red or a heat shock protein significantly attenuated CryAB R120G -induced UPS malfunction in cultured cardiomyocytes. Because of the central role of the UPS in cell regulation and the high intrasarcoplasmic amyloidosis prevalence in failing human hearts, our data suggest a novel pathogenic process in cardiac disorders with abnormal protein aggregation. (Circ Res. 2005;97: ) Key Words: proteasome ubiquitin protein aggregation B-crystallin desmin-related cardiomyopathy amyloidosis transgenic mice Most cellular proteins are degraded through the ubiquitin proteasome system (UPS). UPS-mediated proteolysis includes 2 major steps: attachment of a chain of ubiquitin to the target protein molecule through a process known as ubiquitination and degradation of the ubiquitinated proteins by the 26S proteasome. The latter consists of a barrel-shaped 20S core and the 19S cap on 1 or both ends of the 20S. The actual proteolytic activity resides in the interior of the 20S, whereas the 19S plays a critical role in channeling ubiquitinated protein molecules into the 20S. 1 Ubiquitinated proteins accumulate in the cell when the proteasome is inhibited. 1 Aberrant protein aggregation is a common process in many neural degenerative diseases such as Alzheimer s, Parkinson s, and Huntington s diseases. 2,3 Remaining to be demonstrated in intact animals, this process was shown in cell culture to impair UPS-mediated proteolysis. 4 Therefore, UPS malfunction is considered an important pathogenic mechanism in neurodegeneration. 5 Recent studies revealed that abnormal protein aggregation, intrasarcoplasmic amyloidosis (IA), and accumulation of ubiquitinated proteins are also very common phenomena in human hearts with idiopathic or ischemic cardiomyopathies. 6 9 These observations, especially the discovery that very abundant amyloid-positive substance is strikingly associated with myofibrils in most human hearts with dilated or hypertrophic cardiomyopathies, suggest IA as a potential major pathogenic process in congestive heart failure (CHF). 8 Hence, elucidation of the effect of IA on UPS-mediated proteolysis may provide significant insight into the molecular pathogenesis of CHF. Original received June 27, 2005; revision received September 20, 2005; accepted September 27, From the Cardiovascular Research Institute (Q.C., J.-B.L., K.M.H., H.Z., A.R.K.K., J.L., F.L., A.M.G., X.W.), South Dakota Health Research Foundation, University of South Dakota School of Medicine and Sioux Valley Hospitals and Health System, Sioux Falls; National Eye Institute (E.F.W.), National Institutes of Health, Bethesda, Md; Department of Pathophysiology (J.-B. L), Guangzhou Medical College, Guangzhou, Guangdong, China; and Department of Laboratory Medicine (F. L.) and Division of Basic Biomedical Sciences (Q.C., J.-B.L., K.M.H., H.Z., A.R.K.K., J.L., X.W.), University of South Dakota School of Medicine, Sioux Falls. *Both authors contributed equally to this work. Correspondence to Xuejun Wang, MD, PhD, Cardiovascular Research Institute, University of South Dakota School of Medicine, 1100 E 21st St, Ste 700, Sioux Falls, SD xwang@usd.edu 2005 American Heart Association, Inc. Circulation Research is available at DOI: /01.RES b 1018

2 Chen et al Proteasome Malfunction in Crystallinopathy 1019 A missense mutation (R120G) in the B-crystallin (CryAB) gene encoding a molecular chaperone highly expressed in the heart has been shown to cause aberrant protein aggregation in the heart and result in desmin-related cardiomyopathies (DRC) in humans and transgenic (Tg) mice. 10,11 Mice carrying 3 copies of a Tg consisting of the mouse -myosin heavy chain promoter and the CryAB R120G cdna show no apparent cardiac abnormality at 1 month but develop concentric cardiac hypertrophy with diastolic malfunction at 3 months and die of CHF between 5 and 7 months. 11 It was recently demonstrated that CryAB R120G mice were a model of cardiac IA, resembling a prominent feature in failing human hearts. 8 Therefore, CryAB R120G mice are used as an animal model to study the pathogenesis of aberrant protein aggregation and IA in the present study. We hypothesized that aberrant protein aggregation induced by CryAB R120G impairs UPS proteolytic function in the heart, representing a nodal pathogenic process in cardiac remodeling and failure in DRC. We present here several lines of evidence that clearly support this hypothesis. We have demonstrated in intact animals that expression of a misfolded and aggregation-prone cytosolic protein impairs the proteolytic function of the UPS in the heart before other cardiac pathology becomes discernible. The defect appears to reside in the delivery of ubiquitinated protein molecules into the proteolytic cavity of the 20S proteasomes, and depletion of key components of 19S proteasomes may be responsible. Using cultured cardiomyocytes, we have further proven that aberrant protein aggregation is an essential process for CryAB R120G to induce proteasomal malfunction. Because aberrant protein aggregation and accumulation of ubiquitinated proteins, both indicative of malfunction in protein quality control, have been frequently observed in failing human hearts, protein aggregation-induced UPS malfunction likely represents a potentially important pathogenic process in not only DRC but also other cardiac disorders with protein misfolding. Materials and Methods An expanded Materials and Methods section is available in the online data supplement at Tg Mice FVB/N Tg mice with cardiac-specific overexpression of CryAB R120G or wild-type (WT)-CryAB have been described. 8,11 Generation of CryAB-null mice has been reported previously. 12 FVB/N Tg mice ubiquitously expressing a reporter substrate (GFPdgn) of the UPS are described elsewhere. 13 GFPdgn is created by fusion of degron CL-1 to the carboxyl terminus of the conventional green fluorescent protein (GFP). Previous studies have proven that this modification renders GFP a specific substrate for the UPS and that resultant GFPdgn can serve as a dynamic indicator for the proteolytic function of the UPS in cardiomyocytes. 4,14 All animals used in this study were produced by the Laboratory Animal Facility of the University of South Dakota. Institutional guidelines for the care and use of the animals were followed. In Vitro GFPdgn Degradation Assay To assess the proteolytic function of the entire UPS, GFPdgn was purified from the skeletal muscle of GFPdgn mice by immunoaffinity chromatography (Pierce). Equal amounts of GFPdgn protein were incubated with 10 g of soluble protein from ventricular myocardium. The reactions were performed in 25 mmol/l Tris-HCl buffer containing ATP (2 mmol/l) and MgCl 2 (2 mmol/l) for 30 minutes Figure 1. Ubiquitin conjugates were increased in CryAB R120G Tg (R120G) mouse hearts. Free ubiquitin (Ub) and ubiquitinated proteins in ventricular total protein (A and B) and soluble protein (C and D) extracts from 1-month-old (1m) and 3-month-old (3m) mice were quantitatively analyzed by immunoblots. *P 0.01 compared with Ntg or CryAB Tg (WT-TG); 1-way ANOVA followed by the Scheffe test. AU indicates arbitrary units. in the presence or absence of MG-132 (10 mol/l). The reactions were stopped by adding 3 SDS-PAGE sampling buffer and immediately followed by boiling for 5 minutes. The end product was then fractionated by 12% SDS-PAGE and immunoprobed for GFPdgn. Results Ubiquitinated Proteins Were Progressively Increased in CryAB R120G Mouse Hearts Because cardiac expression of CryAB R120G not only recapitulates manifestations of human DRC but also displays IA that is frequently observed in non-drc failing human hearts, 8,11 CryAB R120G Tg mice serve as a valuable model to dissect mechanisms underlying CHF. To explore the pathogenesis of CryAB R120G, we found that ubiquitinated proteins in both the soluble fraction and total protein extracts from the heart progressively increased in CryAB R120G Tg but not WT-CryAB Tg mice at 1 month and 3 months, whereas free ubiquitin remained unchanged compared with Ntg littermates (Figure 1A through 1D). Ubiquitinated proteins are normally degraded efficiently by the 26S proteasomes. 1 An increase in ubiquitinated proteins indicates either proteasomal malfunction or enhanced ubiquitination. Because the overexpression of CryAB R120G at the transcript level is much less than overexpression of WT-CryAB in these Tg mice, 11 increased ubiquitination resulting from CryAB protein overexpression could not account for the significant difference in ubiquitinated protein levels between CryAB R120G

3 1020 Circulation Research November 11, 2005 Figure 2. The UPS is impaired in CryAB R120G Tg hearts. A through C, At 1 (A) and 3 (B) months, ventricular myocardium was collected from indicated littermate mice resulting from crossbreeding between CryAB R120G and GFPdgn heterozygous Tg mice to determine GFPdgn using quantitative immunoblot analysis. In A and B, a nonspecific band (NS) detected by this anti-gfp antibody was used as the in-lane loading control. kd indicates kilodaltons. Quantitative data from 6 hearts of each group are shown in C. *P 0.01, R120G/GFPdgn TG vs CryAB/GFPdgn TG and GFPdgn TG. D and E, Northern blot analyses of the steady-state GFPdgn transcript levels in the ventricles of 1-month-old (1m) and 3-month-old (3m) GFPdgn Ntg, GFPdgn single-tg, and GFPdgn/R120G double-tg littermates. The 28S rrna was used as loading control for densitometry (E). F, In vitro GFPdgn degradation assays. Equal amount of GFPdgn protein purified from GFPdgn mice was used as the substrate to incubate for 30 minutes with ventricular protein extracts from 3-month-old Ntg, CryAB Tg, or R120G Tg mice in the presence of a proteasome inhibitor (MG-132) or its vehicle (DMSO). The remaining GFPdgn protein was then quantified using Western blots. A reaction containing GFPdgn alone in the buffer was included as the control (Control), and its GFPdgn protein signal was set as 1 arbitrary unit (AU). The remaining GFPdgn protein in other reactions was normalized to the control. A representative immunoblot image (top) is shown, along with the graph (bottom) summarizing the quantitative results from 4 hearts of each group. *P 0.01, compared with corresponding MG-132 treated group; #P 0.05, compared with NTG/DMSO or CryAB/DMSO groups; 1-way ANOVA followed by the Scheffe test. and WT-CryAB Tg hearts. Therefore, these findings suggest that the 26S proteasome is likely malfunctioning in CryAB R120G expressing hearts. UPS Ability to Degrade a Surrogate Substrate Was Progressively Diminished in CryAB R120G Mouse Hearts, Whereas Proteasomal Peptidase Activities Were Not Reduced We have recently established and validated a Tg mouse model in which a reporter substrate (GFPdgn) of the UPS is ubiquitously and constitutively expressed in all the major organs. 13 Changes in GFPdgn protein levels inversely reflect the proteolytic function of the UPS. To probe UPS proteolytic function in CryAB R120G mouse hearts, we crossbred CryAB R120G or WT-CryAB Tg mice with GFPdgn mice. As observed in hearts treated with specific proteasome inhibitors, 13 GFPdgn protein was dramatically increased in CryAB R120G /GFPdgn double-tg hearts at both 1 month and 3 months, compared with GFPdgn single-tg hearts of littermates. GFPdgn protein was not increased in WT-CryAB/ GFPdgn double-tg hearts (Figure 2A through 2C). Consistently, direct fluorescence confocal microscopy of myocardium showed clearly increased green fluorescence intensities in the cardiomyocytes of CryAB R120G /GFPdgn double-tg hearts (Figure 3). The increase in GFPdgn protein in CryAB R120G /GFPdgn double-tg hearts was greater at 3 months than at 1 month (Figures 2C and 3). This increase in GFPdgn protein is attributable to decreased degradation rather than increased synthesis, because the steady-state GFPdgn transcript level was not increased in CryAB R120G / GFPdgn double-tg hearts compared with GFPdgn single-tg hearts (Figure 2D and 2E). Furthermore, the ability of crude protein extracts from CryAB R120G hearts to degrade in vitro GFPdgn protein immunoprecipitated from the skeletal muscle of GFPdgn mice was significantly compromised compared with Ntg littermates or WT-CryAB Tg controls (Figure 2F). These tests prove that cardiac UPS proteolytic function is significantly impaired by expression of CryAB R120G. The free ubiquitin levels were not altered. Moreover, ubiquitinated proteins were significantly increased in CryAB R120G hearts (Figure 1), suggesting that the primary cause of UPS malfunction in these hearts is not at ubiquitin conjugation but rather in the 26S proteasome. The latter is composed of the 19S and the 20S subcomplexes. The 19S is believed to recognize ubiquitinated protein molecules, deubiquitinate and unfold them, and channel the unfolded protein to the 20S, where actual proteases reside. The 20S proteasome is a hollow cylindrical protein complex composed of 2 central antiparallel rings flanked by 2 identical rings. Each or ring consists of 7 protein molecules ( 1 to 7 and 1 to 7 ). The unfolded protein is believed to be degraded in the cavity of the 20S by 3 major peptidase activities: chymotrypsin-like, trypsin-like, and caspase-like (also known as peptidylglutamyl-peptide hydrolase) activities, which reside in the 5, 2, and 1 subunits, respectively. 1 We measured these peptidase activities using synthetic fluorogenic substrates and crude protein extracts from the heart. WT-CryAB Tg mice do not show any abnormal phenotype,

4 Chen et al Proteasome Malfunction in Crystallinopathy 1021 Figure 3. Direct green fluorescence confocal micrographs. Cryosections from perfusion-fixed ventricular myocardium of 1-month-old (A) or 3-month-old (B) mice resulting from crossbreeding between CryAB R120G and GFPdgn heterozygous Tg mice were examined with a confocal microscope. In both A and B: a, Ntg; b, CryAB single-tg; c, CryAB/GFPdgn double-tg; d, GFPdgn single-tg; e, R120G single-tg; f, R120G/GFPdgn double-tg. The confocal imaging system was set up to allow the low-level background green fluorescence in the GFPdgn Ntg hearts to be captured to visualize the outline of cardiomyocytes in each section. The green fluorescence intensity in the GFPdgn single-tg hearts (A, d; B, d) is not appreciably greater than that in the Ntg hearts (a, b, and e in A and B) because this GFPdgn Tg line (line 2) has relatively low GFPdgn protein expression in the heart. The background green fluorescence in the R120G single-tg heart at 3 months (B, e) appears to be greater than Ntg littermates. Nevertheless, GFPdgn fluorescence intensities were markedly increased in R120G/GFPdgn double-tg hearts (f in A and B) but not in CryAB/GFPdgn double-tg hearts (c in A and B) at 1 and 3 months. The accumulation of GFPdgn protein in R120G/GFPdgn double-tg hearts occurred in cardiomyocytes, and, especially at 3 months, it tends to be in an aggregated form (B, f). Scale bar 20 m. AU indicates arbitrary units. and the UPS proteolytic function in their hearts does not differ from Ntg littermates (Figures 1 through 3). Therefore, for brevity, WT-CryAB Tg mice were not included in studies described in Figures 4 and 5. Compared with Ntg littermates, all 3 forms of peptidase activities were, surprisingly, not decreased in CryAB R120G hearts at 1, 3, or 6 months. On the contrary, trypsin-like activities were significantly increased at all of the 3 time points, especially at 3 and 6 months, and the chymotrypsin-like and the caspase-like activities were also significantly increased at 6 months when CHF becomes overt (Figure 4). These findings suggest that the impairment of Figure 4. Changes in the peptidase activities of 20S proteasomes. In vitro peptidase assays using synthetic fluorogenic peptides (Calbiochem and Biomol) as substrates revealed that chymotrypsin-like (A) and caspase-like (B) activities were not significantly changed at either 1 month (1m) or 3 months (3m), but they were significantly increased at 6 months (6m) in the CryAB R120G Tg (R120G-TG) hearts compared with Ntg littermates. Trypsin-like activities (C) were significantly increased at all the 3 time points. *P 0.01, **P 0.05, CryAB R120G Tg vs Ntg; Student s t test. AU indicates arbitrary units. proteasomal proteolytic function in CryAB R120G hearts is attributable to a defect in the uptake of ubiquitinated proteins into 20S proteasomes rather than altered peptide cleavage activities of the 20S. It is generally accepted that the 19S

5 1022 Circulation Research November 11, 2005 Figure 5. Changes in the abundance of key components of 19S and 20S proteasomes. Total protein extracts from the ventricles of CryAB R120G Tg (R120G-TG) and Ntg littermates were fractionated by SDS-PAGE and analyzed quantitatively using Western blots for the indicated subunits. Western blot images and densitometry data are shown in A and B, respectively. At 1 month (1m), the Rpt3 and Rpt5 subunits of the 19S and the 6 (20S- 6), 2 (20S- 2), and matured 5 (the lower band of 20S- 5) subunits of the 20S remained unchanged, whereas the 5 precursor (boxed in A; pr- 5 inb) of the 20S was significantly increased. At both 3 months (3m) and 6 months (6m), however, Rpt3 and Rpt5 were significantly decreased, whereas all of the examined subunits of the 20S were significantly increased in R120G Tg hearts. *P 0.01, **P 0.05, compared with Ntg; Student s t test; n 3. AU indicates arbitrary units. proteasome and its interaction with the rings of the 20S regulate and mediate the entry of targeted protein molecules into the 20S. 15 Consistently, the protein abundance of the 20S components ( 6, 2, 5, and the precursor of 5 ) was significantly increased, whereas key components (Rpt3 and Rpt5) of the 19S evidently decreased in CryAB R120G hearts at 3 and 6 months (Figure 5). Aberrant Protein Aggregation Plays an Essential Role in UPS Impairment by CryAB R120G CryAB R120G is misfolded and aggregation prone. Its presence dominantly inhibits the chaperone function of normal CryAB. 16,17 To ascertain whether UPS impairment by CryAB R120G is caused by loss of function of CryAB, we examined UPS functional status in CryAB-null mouse hearts at 2 to 3 months, when no discernible phenotype is evident. Compared with age-matched wild-type mice of the same genetic background, neither the abundance of ubiquitinated proteins nor proteasomal peptidase activities were significantly altered in the CryAB-null hearts (data shown in the online data supplement). This suggests that UPS malfunction observed in CryAB R120G expressing hearts is not caused by loss of function of the CryAB gene. Because CryAB can oligomerize with other small heat shock proteins (Hsp) and may function in heterooligomers, 17 the results from CryABnull mice do not rule out a possible contribution of the dominant negative activity of CryAB R120G to the UPS malfunction. In cultured HEK cells, aberrant protein aggregation of mutant huntingtin and cystic fibrosis membrane conductor protein was shown to be sufficient to impair the UPS. 4 Adenovirus-mediated overexpression of truncated cardiac myosin-binding protein C mutants were recently shown to impair the UPS in cultured NRVMs and form abnormal aggregates, but a causal relationship between aberrant protein aggregation and UPS impairment was not tested. 18 We have further addressed these critical issues in a well-established NRVM culture system. CryAB R120G, WT-CryAB, and a UPS function reporter (GFPu or GFP u ) similar to GFPdgn were introduced into cultured NRVMs via adenovirus infection. We reported that changes in GFPu protein levels in cultured NRVMs inversely reflect the proteolytic function of the UPS. 14 As observed in Tg mouse hearts, expression of CryAB R120G but not WT-CryAB caused significant increases of GFPu protein in cultured NRVMs in a dose-dependent manner (Figure 6A). The increase in GFPu protein levels marks a decrease in UPS proteolytic function because the synthesis of GFPu, as demonstrated by steady-state GFPu transcript levels, was not increased by coexpression of CryAB R120G compared with coexpression of either WT- CryAB or -galactosidase (Figure 6B). These findings demonstrate that modest overexpression of CryAB R120G, but not

6 Chen et al Proteasome Malfunction in Crystallinopathy 1023 Figure 6. Expression of CryAB R120G impairs the proteasome in cultured cardiomyocytes. A, UPS-specific surrogate substrate GFPu was expressed in NRVMs via adenovirus (Ad-GFPu) infection. Coexpression of CryAB R120G (R120G), but not WT-CryAB (CryAB) or -galactosidase ( -Gal), caused accumulation of GFPu protein in NRVMs in a dose-dependent manner. B, Steady-state transcript levels of GFPu were analyzed with Northern blots. Compared with coexpression of CryAB or -galactosidase ( -Gal), coexpression of CryAB R120G did not increase GFPu transcript levels. C and D, Western blot analyses of ubiquitinated (Ubed) proteins and free ubiquitin (Ub) in cultured NRVMs. A representative Western blot image is shown in C, and quantitative data from 4 independent repeats are summarized in D. Compared with the group without adenoviral infection (No-Ad), Ub remained unchanged, whereas ubiquitinated proteins were significantly increased in cells overexpressing CryAB R120G (R120G) but not CryAB. *P 0.01, R120G vs CryAB or No-Ad; 1-way ANOVA followed by the Scheffe test. AU indicates arbitrary units. WT-CryAB, is sufficient to impair the UPS in cultured cardiomyocytes. Similar to what was observed in the heart, the abundance of free ubiquitin was not affected; however, the ubiquitinated proteins were significantly increased by CryAB R120G but not WT-CryAB overexpression in cultured NRVMs (Figure 6C and 6D). These findings indicate that changes in ubiquitin conjugation do not likely contribute to the UPS proteolytic malfunction in CryAB R120G -expressing cells. This is consistent with in vivo findings and implicates that the primary defect is in the proteasome. The major pathological change in CryAB R120G -expressing human and mouse hearts and in CryAB R120G -expressing NRVMs is aberrant protein aggregation. CryAB-positive abnormal protein aggregates were observed in CryAB R120G but not in WT-CryAB Tg hearts. 11 In addition to relatively large and microscopically visible aggregates, SDS-soluble high molecular weight CryAB oligomers, which are unable to pass through the nitrocellulose membrane with a pore size of 0.25 m, were significantly increased in the CryAB R120G hearts (Figure 7A, a) and CryAB R120G -expressing NRVMs (Figure 7B). It has been well documented that molecular chaperones, such as Hsp and certain pharmacological agents (eg, Congo red), can bind misfolded proteins and effectively prevent them from aggregating, thereby assisting protein quality control in the cell To test the necessity of abnormal protein aggregation in UPS impairment by CryAB R120G, we determined the effect of reduction of protein aggregation by Congo red treatment or by overexpressing inducible Hsp70 in cultured NRVMs. Interestingly, as shown by the nitrocellulose filter-trapping assay, the prevalence of CryAB oligomers in the myocardial homogenate from CryAB R120G hearts was dramatically reduced by in vitro treatment of Congo red (Sigma) in a dose-dependent manner (Figure 7A, b). Consistent with a previous report, 8 the treatment of Congo red on NRVMs in primary culture significantly reduced the formation of both the microscopically visible protein aggregates (Figure 7D) and the soluble CryAB oligomers induced by expression of CryAB R120G (Figure 7B and 7C). Importantly, this inhibition of aberrant protein aggregation by Congo red led to a significant attenuation of UPS malfunction, as evidenced by a significantly decreased GFPu accumulation (Figure 7B through 7D). Similar reduction in both CryAB oligomers and GFPu accumulation was also observed with coexpression of inducible Hsp70 (data not shown). These findings prove that aberrant protein aggregation plays an essential role in CryAB R120G induced proteasomal malfunction. Discussion UPS-mediated proteolysis degrades abnormal proteins such as misfolded, oxidized, and mutant proteins, thereby serving as a critical quality control in the cell. The UPS also degrades normal proteins that are no longer needed. This regulatory degradation ensures timely removal of normal proteins, including signaling proteins and transcription regulators such as mitotic cyclins, CDK inhibitors, I B, -catenin, and calcineurin when their jobs are done. 1,23 Therefore, the UPS plays critical roles in virtually all important cellular pro-

7 1024 Circulation Research November 11, 2005 Figure 7. Reduction of aberrant protein aggregation by Congo red attenuates CryAB R120G induced UPS impairment. A, Filter-trap assays for high molecular weight CryAB polymers. a, Shown is a marked increase of SDS-resistant CryAB polymers in the R120G heart compared with the Ntg or CryAB Tg heart. b, In vitro incubation (90 minutes) of Congo red dissolved in PBS with the protein exacts from R120G hearts reduced CryAB polymers in a dose-dependent manner. The control is the same protein extract incubated with PBS for the same length of time as the Congo red incubation. B through D, Inhibition of protein aggregation by Congo red attenuates R120G induced UPS impairment in cultured NRVMs. Representative Western blot images are shown in B. Oligo CryAB indicates CryAB protein oligomers. C, Summary of quantitative data of 4 independent repeats of experiments shown in B. *P 0.05, **P 0.01, R120G/CR vs R120G. CR indicates Congo red. D, Representative fluorescence micrographs illustrating that expression of CryAB R120G causes formation of CryAB aggregates (a, red) and accumulation of GFPu (b, green), but Congo red treatment (c and d) blocked aberrant CryAB aggregation (c) and attenuated GFPu accumulation (d). c and d, Congo red treated; a and b, the untreated controls. CryAB immunofluorescence (red) images (a and c) and images of their overlay with GFPu direct green fluorescence (b and d) are shown. Scale bar 2.5 m. AU indicates arbitrary units. cesses, such as cell-cycle control, transcription regulation, signal transduction, and cell survival and homeostasis. Until very recently, the elucidation of the involvement of UPS dysfunction in pathogenesis of disease has been hindered by a lack of reliable methods to measure dynamic changes of UPS function in vivo. Recently established UPS functional reporter Tg mouse models make it possible to monitor in vivo dynamic changes in the proteolytic function of the entire UPS. 24 Taking advantage of 1 such mouse model that we have recently created and validated, 13 we have collected in the present study definitive in vivo evidence that modest cardiac overexpression of a human DRC-linked mutant CryAB impairs the proteolytic function of the UPS in the heart. This impairment occurred before cardiac hypertrophy, and malfunction became discernible. Cardiac remodeling and malfunction progressed as UPS function in the heart deteriorated progressively, suggesting that UPS malfunction likely participates in the pathogenesis of the mutant CryAB. Further analyses reveal that the UPS malfunction is mainly attributable to a defect in the delivery of ubiquitinated proteins into the proteolytic chamber of the 20S proteasome, and the depletion of key components of the 19S proteasome may be responsible. Because of accumulation of ubiquitinated proteins and alteration of proteasomal peptidase activities were not evident in CryAB-null mice, proteasomal malfunction in CryAB R120G Tg hearts is unlikely caused by loss of function of the CryAB gene. Our findings from experiments with cultured NRVMs further suggest that aberrant protein aggregation is a major underlying mechanism by which misfolded CryAB causes proteasomal malfunction. Tg Expression of CryAB R120G Impairs the Delivery of Ubiquitinated Proteins Into 20S Proteasomes By crossbreeding UPS functional reporter (GFPdgn) mice with CryAB R120G Tg mice, we are able to prove that proteolytic function of the UPS is severely impaired in CryAB R120G Tg hearts (Figures 1 through 3). However, conventional proteasomal peptidase activity assays failed to detect the impairment. On the contrary, all of the 3 peptidase activities showed significant increases in the CryAB R120G heart at the CHF stage (Figure 4). These peptidase assays use synthetic fluorogenic substrates, which are small peptides and can easily diffuse into the proteolytic chamber of the 20S proteasome; therefore, they only measure the function of the 20S proteasomes. 25 In the CryAB R120G heart, ubiquitin conjugation does not appear to be a problem because ubiquitinated

8 Chen et al Proteasome Malfunction in Crystallinopathy 1025 proteins are progressively increased (Figure 1A through 1D). The function of 20S proteasomes is unlikely to be the primary cause of observed proteasomal malfunction because conventional in vitro assays showed that their peptidase activities are not reduced at all but rather significantly increased at the CHF stage (Figure 4). These analyses suggest that the primary defect likely resides at the transport of ubiquitinated proteins to the 26S proteasome and the entry of ubiquitinated proteins into the 20S proteasome. The process by which target proteins are delivered into the proteolytic core of the 20S proteasome is incompletely understood. Although other proteins, such as the CDC48 protein complex, may be involved, 26 it is generally believed that 19S proteasomes play a primary role in this process. 1,15 Therefore, we assessed the abundance of key components of the 19S and the 20S proteasomes and discovered that Rpt3 and Rpt5 of the 19S were significantly decreased, whereas all of the examined subunits of the 20S were markedly increased in CryAB R120G hearts at 3 and 6 months, when overt cardiac hypertrophy and malfunction were evident (Figure 5). It was reported that inhibition of proteasome activity in cultured mammalian cells induces concerted expression of proteasome genes including components of the 19S and the 20S and de novo formation of the 26S proteasome. 27 This is considered a compensatory response to acquired proteasome malfunction. Hence, in CryAB R120G hearts, which clearly show proteasome malfunction, the upregulation of 20S proteasome components is likely a compensatory response, whereas the depletion of 19S proteasome components is probably a cause for the deficiency in the delivery of ubiquitinated proteins into the 20S. It is also worthwhile to note that discoveries presented here also illustrate that it can be very misleading to use proteasomal peptidase activity assays alone to evaluate the proteolytic function of the UPS or the proteasome. UPS Impairment by Aberrant Protein Aggregation Is Potentially a Novel Pathogenic Process for Cardiac Remodeling and Failure This postulate remains to be further tested, but it is consistent with multiple lines of direct and indirect evidence. First, aberrant protein aggregation is not just a feature for DRC but is rather a common phenomenon in human CHF. It was recently discovered that IA (a form of aberrant protein aggregation) from unknown proteins was frequently present in the cardiomyocytes of failing human hearts with either hypertrophic or dilated cardiomyopathies. 8 Aberrant protein aggregation was also observed in ischemic cardiomyopathy and CHF resulting from dilated cardiomyopathy. 6 Second, proteasomal malfunction has been implicated in failing human hearts. Immunohistologic studies of failing human hearts revealed that increased ubiquitin conjugates were colocalized with autophagic cell death, an important form of cell death in the failing heart. 7 It was also reported that ubiquitinated protein levels were significantly increased in the heart with either dilated or ischemic cardiomyopathies, 9 suggesting that proteasomal function is likely impaired in failing human hearts. Third, studies with cell culture have previously shown impairment of the UPS by aberrant protein aggregation. 4,18 The present study proves that UPS proteolytic function is severely impaired by expression of an aggregation-prone protein in the heart of intact mice. Our additional tests with cultured NRVMs demonstrate that aberrant protein aggregation is not only sufficient but also essential for a mutant CryAB to impair UPS function. Although it has not been tested whether UPS malfunction causes or potentiates cardiac hypertrophy and/or failure, it has been reported that several signaling proteins, such as -catenin and calcineurin, 1,23 which mediate cardiac growth, including pathological hypertrophy, are degraded by the UPS. Indeed, we found that cytosolic -catenin was significantly increased in CryAB R120G Tg hearts at the CHF stage (supplemental Figure I). Activated calcineurin was previously found to be markedly increased in failing human hearts. 28 It will be very important to determine whether the increase in these signaling proteins in CHF is attributable to a decrease in their degradation. Finally, proteasome inhibition leads to apoptosis, a major form of cell death that is considered an important mechanism underlying CHF. 29 Cells, at least proliferating cells, defective in the UPS most often arrest near the G 2 /M boundary of the cell cycle and undergo apoptosis. 30 We have previously observed that proteasomal inhibition by MG-132 increased cell death in cultured NRVMs. 14 It has not been reported but should be extremely important to ascertain whether proteasome malfunction affects cardiomyocyte survival in vivo. Sarcomeres are the most fundamental function units of cardiac contractility. Normal turnover of most sarcomeric proteins depends on the UPS. 31,32 Failure to replace aged or damaged sarcomeric proteins would conceivably afflict the behavior and performance of myofibrils. Furthermore, the endoplasmic reticulum (ER) plays critical roles in the quality control of membrane proteins and proteins for secretion. The ER accomplishes this important task in partnership with the UPS. In fact, the proteasome is believed to be responsible for the degradation of all abnormal cellular proteins, thereby executing quality control in the cell. 1 Prolonged ER stress has been reported as a mechanism underlying heart failure. 33 It would not be surprising if UPS malfunction played an even greater role than prolonged ER stress in cardiac remodeling and failure. It should be pointed out that the UPS is the major, but by no means the only, proteolytic pathway responsible for protein turnover in cardiomyocytes. 32 Other pathways, such as the calpain proteolytic pathway and the lysosomal, autophagic pathway, also exist and likely play a role in cardiomyocytes. 34,35 It is unknown but should be very interesting to investigate whether these other pathways compensate for UPS proteolytic deficiency. Acknowledgments This work was supported by NIH grants HL72166 (to X.W.) and P20 RR (to A.M.G., X.W., and F.L.) and American Heart Association Scientist Development Grant N (to X.W.). A.R.K. is supported by a Predoctoral Fellowship Grant ( Z) from the American Heart Association. We thank Dr Jeffrey Robbins for generously providing the WT-CryAB and CryAB R120G Tg mouse models. We also thank the Cell Culture Core in the Cardiovascular Research Institute of the University of South Dakota for technical assistance in NRVM culture.

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